Cathepsin L activity is essential to elastase perfusion-induced abdominal aortic aneurysms in mice

Abstract

Objective: The development of abdominal aortic aneurysms (AAA) requires extensive aortic wall matrix degradation. Human AAA lesions express high levels of cathepsin L (CatL), one of the most potent mammalian elastases. Whether this protease participates directly in AAA pathogenesis, however, is unknown.

Methods and results: We generated experimental AAA with aortic elastase perfusion in mice and established an essential role of CatL in AAA formation. After 14 days postperfusion, most wild-type (Ctsl(+/+)) mice developed AAA, but none of the CatL-deficient (Ctsl(-/-)) mice did. AAA lesion macrophage contents, CD4(+) T cell numbers, CD31(+) and laminin-5 angiogenic fragment γ2(+) microvessel numbers, and elastin fragmentation were all significantly lower in Ctsl(-/-) mice than in Ctsl(+/+) mice. While lesions from Ctsl(-/-) mice contained fewer Ki67(+) proliferating cells than did Ctsl(+/+) mice, the absence of CatL did not affect lesion apoptotic cell contents or medial smooth-muscle cell loss significantly. Mechanistic studies indicated that the absence of CatL reduced lesion chemokine monocyte chemotactic protein-1 content, macrophage and T-cell in vitro transmigration, and angiogenesis, and altered the expression and activities of matrix metalloproteinases and other cysteinyl cathepsins in inflammatory cells, vascular cells, and AAA lesions.

Conclusion: CatL contributes to AAA formation by promoting lesion inflammatory cell accumulation, angiogenesis, and protease expression.

Figure 1

Aortic expansion in elastase perfusion-induced experimental abdominal aortic aneurysms. Aortic diameters from Ctsl^+/+ mice and Cts^–/– mice at different time points: preperfusion, immediate postperfusion (day 0), and at harvesting on day 7 (A) and day 14 (B). Data are mean±SE. The number of mice per group is indicated in each bar. P<0.05 was considered statistically significant, 2-way repeated-measures ANOVA test followed by Bonferroni posthoc correction. NS indicates not significant.

Figure 2

Abdominal aortic aneurysms (AAA) lesion inflammatory cell accumulation. Mac-3⁺ macrophages (A) and CD4⁺ T cells (B) accumulated less in AAA lesions from Ctsl^–/– mice than in those of Ctsl^+/+ mice at 14 days postperfusion. At the same time point, MCP-1⁺ cell numbers were also fewer in AAA lesions from Ctsl^–/– mice than in Ctsl^+/+ mice (C). Data are mean±SE. The number of mice per group is indicated in each bar. P<0.05 was considered statistically significant; Mann-Whitney U test. Representative images are shown to the right in each panel. All measurements are from the entire lesion including both adventitia and media.

Figure 3

Cathepsin L function in neovascularization in abdominal aortic aneurysms (AAA) lesions. CD31⁺ (A) and proangiogenic laninin-5 fragment γ2⁺ (B) microvessel numbers were reduced in AAA lesions from Ctsl^–/– mice. The number of mice per group is indicated in each bar. Both measurements are from the entire lesion including adventitia and media. Aortic ring assay in vitro demonstrated impaired microvessel sprouting from Ctsl^–/– mouse aortic rings with or without angiogenic factor bFGF (C). Representative images for panels A–C are shown to the right. RT-PCR showed reduced transcripts of cathepsins B and K, and matrix metalloproteinases (MMP)-2 in microvessel endothelial cells (MHEC) from Ctsl^–/– mice compared with those from Ctsl^+/+ mice (D). Cysteine protease active site labeling with JPM (E) and gelatin gel zymogram (F) demonstrated impaired cathepsin and MMP activities in MHEC from Ctsl^–/– mice, respectively. GAPDH immunoblot was used for protein loading control. All data are mean±SE. P<0.05 was considered statistically significant; Mann-Whitney U test.

Figure 4

Cathepsin L deficiency and abdominal aortic aneurysms (AAA) lesion cell proliferation and apoptosis. AAA lesion Ki67⁺ proliferating cells were reduced in Ctsl^–/– mice at 7 days and 14 days postperfusion (A; representative images are shown to the right), but cathepsin L deficiency did not affect AAA lesion cell apoptosis (B) or lesion medial smooth-muscle cell loss (C). The number of mice per group is indicated in each bar. Measurements in panels A and B are from the entire lesion including adventitia and media. All data are mean±SE. P<0.05 was considered statistically significant; Mann-Whitney U test.

Figure 5

Medial elastin fragmentation and protease expression. A, Medial elastin fragmentation was reduced in abdominal aortic aneurysms (AAA) lesions from Ctsl^–/– mice. The number of mice per group is indicated in each bar. B, RT-PCR demonstrated altered cathepsin and matrix metalloproteinases (MMP) transcript levels in aortic smooth-muscle cell from Ctsl^–/– mice compared with those of Ctsl^+/+ mice. All data are mean±SE. P<0.05 was considered statistically significant; Mann-Whitney U test. C, In situ fluorescent zymogram assay showed high elastinolytic cysteinyl cathepsin activities in the adventitia of Ctsl^+/+ mouse AAA lesions (top left) that can be largely inhibited by 20 μmol/L of E64d (top right). Such activities were reduced in AAA lesion adventitia from Ctsl^–/– mice (bottom left), and E64d further reduced cathepsin activities (bottom right). The percentages of adventitia fluorescence yielded from fluorogenic elastin degradation represented cysteinyl cathepsin activities. The white dotted lines separate medial elastin autofluorescence from the adventitia. D, Immunohistological analysis showed reduced medial expression of cathepsin S (top panels) and cathepsin K (bottom panels) in AAA lesions from Ctsl^–/– mice, compared with those from Ctsl^+/+ mice.

Figure 6

Periaortic CaCl₂ injury-induced abdominal aortic aneurysms (AAA). A, Aortic diameter increase in percentage at 4 weeks after injury. B, Medial elastin fragmentation grades, as determined according to previously published keys (Ref. 19). Representative figures are shown to the right. Data are mean±SE. P<0.05 is considered statistically significant, Mann-Whitney U test. The number of mice for each group is indicated in the bar.